This proposal is in response to the small grants program for investigators coming into vision research for the first time; the PI's background is in molecular parasitology. Opportunistic infection of the cornea with Acanthamoeba spp. causes a chronic, severe keratitis which is refractory to medical treatment and which may be increasing among contact lens users. Development of effective procedures for prevention and treatment of amoebic keratitis has been hampered because of limited knowledge both about the amoeba species involved and their interactions with corneal cells. Construction of defined in vitro systems which contain both amoeba and host cells may allow the development of quantitative and uniform assays for amoeba-host interactions. Defined strains of amoeba isolated from human eye infections will be incubated with permanent corneal epithelial cell cutlures and with primary corneal organ cultures. Several previously developed assay systems which are established in adjacent laboratories will be evaluated for their ability to specifically monitor damage to amoeba and host cells. These include several isotope release assays and several assays based on the microscopic examination of live and fixed amoeba and host cells. Data from the in vitro assay systems will be compared and evaluated with previously published studies on the pathology of amoeba infections of rodent and human corneas. The primary initial goals will be to develop rapid, reproducible in vitro conditions and reliable microscopic or isotopic assay systems. Manipulations of these systems may prove useful in studying the pathogenesis of amoebic keratitis and to screen for effective drug and immune theraphies for amoebic keratitis.